Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site.

Heterotrimeric G proteins (Gαβγ) are molecular switches that alternate between "on" (GTP-bound) and "off" (GDP-bound) states to control the flow of signals from the extracellular milieu to the cytoplasm. Gαs is a prototypical Gα subunit of heterotrimeric G proteins. When bound to GTP, Gαs promotes the synthesis of cAMP in the cytosol through stimulation of the enzymatic activity of adenylyl cyclases. Dysregulation of this mechanism leads to human diseases. For example, naturally occurring mutations like R201C lock Gαs in an active conformation that leads to signaling hyperactivity in cancer. Despite the critical role of Gαs in signaling and its involvement in disease, there are currently no chemical or peptide tools to specifically inhibit its signaling activity. We reasoned that peptides that bind to active Gαs and block the effector binding site would inhibit its ability to signal through adenylyl cyclases. For this, we screened peptides from a phage display library and identified two unrelated peptides that bound to active Gαs. These peptides bound efficiently to GTP-bound Gαs or to the constitutively active Gαs R201C mutant, but not to inactive GDP-bound Gαs. Fluorescence polarization experiments revealed an equilibrium dissociation constant (KD ) of approximately 1 μM for the binding of these peptides to Gαs-GTP. Mutations in the adenylyl cyclase binding region of Gαs greatly reduced binding of these two peptides, indicating that they may work as competitive inhibitors of adenylyl cyclase stimulation. Consistently, both peptides blocked Gas-mediated stimulation of adenylyl cyclase activity in vitrowith μM potency, but had no effect on Gαs-independent stimulation of adenylyl cyclase activity by forskolin. Protein-protein binding experiments revealed that one of the two peptides bound to Gαs but not to Gα subunits of other G protein families, whereas the second peptide bound to Gαi-GTP in addition to Gαs-GTP. In summary, we have identified a peptide inhibitor specific for Gαs-GTP that acts by binding to its effector binding site and precluding adenylyl cyclase stimulation. The peptide identified here could be leveraged as a genetically-encoded probe or derivatized as a cell-penetrating peptide to inhibit Gαs-mediated cell signaling. © FASEB.

Citation

Alex Luebbers, Marcin Maziarz, Mikel Garcia-Marcos. Identification of Peptide Inhibitors of Gαs that Block its Effector Binding Site. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2022 May;36 Suppl 1

PMID: 35556627

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